Plant Regeneration from Cotyledon Protoplasts of Tomato (Lycopersicon esculentum L.)

otyledon protoplasts of tomato (Lycopersicon esculentum L.) isolated from 2–3 week grown seedlings were cultured in MS liquid medium (2,4-D 1, 6-BA 0.1 mg/l) and fresh medium added subsequently. After 6 weeks culture, the cell clusters were transferred to semisolid medium (the additive same as in liquid medium, agar 0.3%). When the calli grew to 0.5 cm in diameter, transfer them to MS medium (6-BA 2, IAA 0.2 mg/l) for differentiation. The regenerated plants were obtained. After comparing different culture methods, tomato protoplasts grew better in double layers than in agar plate and hanging drops.     

Plant Regeneration from Protoplasts of Suspension Cells of Saposhnilkovia divaricata (Turcz.) Schischk

Embryogenic calli were produced from the segments of the young roots, hypocotyls or petioles of test-tube seedlings on MS agar medium containing 1 mg/1 2,4-D. When shaken in the MS liquid medium, the calli formed cell suspension with many embryogenic cell clumps.Using the enzyme mixture: Onozuka R-10 1.5%+MacerozymeR-10 0.3%+Snailase 0.5%+CaCl2 5 mmol/l + Mannitol 0.6 mol/1 (pH=5.8), protoplasts were obtained from the cell clumps which had been subcultured for three to' seven days. When cultivated, the protoplasts grew and began to divide after four days, and formed cell clumps about l—2 mm within fifty days. Protoplast-derived calli were formed from the cell clumps on the MS agar medium with 0.5 mg/l 2,4-D. When transferred onto the MS agar medium containing 0.1 mg/1 6-BA or 0.1 mg/1 2,4-D and 0.5 mg/1 6-BA, the calli differentiated into embryoids. On the MS agar medium without phytohormone, the embryoids grew into plantlets.     

Plant Regeneration from Protoplasts of Peucedanum Terebinthaceum (Fisch.) Fisch. ex Turcz

Calll with many embryogenic cell colonies were produced from segments of seedlling of Peucedanum terebinthaceum (Fisch.) Fisch. ex Turcz. which were cultured on the 1/2MS agar medium (with half quantity of macronutrients) containing 1 mg/l 2,4-D. Cell suspension culture with high percentage of embryogenic cell colonies was established from the calli shaking in liquid medium. The cell suspension culture was used for protoplast preparation. Protoplasts were obtained with the enzyme mixture containing 1.5% Onozuka R-10, 0.3% Macerozyme R-10, 0.5% Snailase, 5 mmol/l CeCl2, 1 mmol/l KH2PO4, 0.6 mol/l mannital at pH 5.8 and 25℃. Cultured in a modified MS liquid medium containing 1 mg/l 2,4-D+ 0.5 mg/l zeatin, the protoplasts emered division after four days, and formed cell colonies of 0.5–1mm after about forty days. When transfered to 1/2 MS liquid medium supplemented with zeatin (0.5 mg/l), the cell colonies differentiated in to embryoids, then developed into plantlets with many green leaves and roots on the 1/2 MS agar medium devoid of phytohormones.     

Plant regeneration from protoplasts of Japanese lawngrass

Embryogenic callus of Japanese lawngrass (Zoysia japonica Steud.) was induced from sterile mature seeds on LS medium with 5 mg / l of 2,4-D. Embryogenic callus selected visually under microscope was proliferated in liquid N6 medium with amino acids (N6-AA medium). Protoplasts were isolated from suspension cells by the treatment of enzyme mixture containing pectolyase Y-23 and cultured in K8p medium with 2 mg / l of 2,4-D at the density of 10⁶ / ml. Plants were regenerated by transferring the protoplasts derived callus to MS medium and incubating at 28 °C under light for two months. Plantlets were successfully transplanted in the soil.Abbreviations 2,4-D 2,4-dichrolophenoxyacetic acid - MES 2-(N-Morpholino) ethanesulfonic acid     

Plant regeneration from meristem-derived callus protoplasts of apple (Malus3domestica cv. `Fuji')

A procedure has been established for regeneration from meristem-derived callus protoplasts of scion cultivars of apple that have been difficult to regenerate from leaf protoplasts. Calli were induced from the meristem of apples, Malus×domestica cvs `Fuji' and `Jonagold' and Malus prunifolia var `ringo Asami Mo84-A', cultured on MS medium (2 mg/l 2,4-D, 1 mg/l BA, 0.8% agar) and subcultured in a liquid medium. The ability to regenerate plants from suspension calli was studied under eight different combinations with respect to IAA, ABA, and TDZ concentrations. With the materials studied here, two combinations, one with 0.1 mg/l IAA, 0.1 mg/l ABA, and 2.0 mg/l TDZ and another with 0.1 mg/l IAA, 1.0 mg/l ABA, and 2.0 mg/l TDZ, were effective for plant regeneration. Protoplasts were isolated from the above suspension cultures and then cultured in KM8P medium containing IBA (2 mg/l), BA (1 mg/l), 2,4-D (0.4 mg/l), and MES (5 mM, pH 5.7). Shoot formation of protoplast-derived calli was studied in the above-mentioned regeneration media. The high concentration of Gelrite (0.5% and 0.7%) was also shown to be important for shoot formation of protoplast-derived calli. Shoot primordia were formed in the medium containing IAA (0.1 mg/l), ABA (1.0 mg/l), and TDZ (2.0 mg/l). Ultimately, five regenerants of `Fuji' protoplasts were obtained from 200 protoplast-derived calli. Received: 19 June 1998 / Revision received: 9 October 1998 / Accepted: 27 October 1998     

Plant Regeneration from Protoplast of Peucedanum praeruptorum Dunn

Calll were initiated from the seedling segment of Peucedanum praeruptorum Dunn and subcultured on the MS agar medium with 0.5 mg/L 2,4-D. Cell suspension culture with a lot of embryogenic cell clumps was obtained in liquid medium. Protoplasts were isolated from the cell clumps in enzyme mixture solution containing 1.5% Onozuka R-10, 0.3% Macerozyme R-10, 0.5% helicase, 5 mmol/L CaCl2 and 0.6 mol/L mannital, at pH 5.6 and shaking for 5- hours at 25℃. Helicase is necessary for isolation. After purified by washing, the protoplasts were cultured in liquid medium containing 1 mg/L 2,4-D +0.5 mg/L zeatin. First cell division was observed after four days. Large cell clumps were formed after thirty days. Microcalli of 1 mm in size was formed after about fifty days, and continued to grow on the MS solid medium containing 0.5 mg/L 2,4-D and 200 mg/L casein hydrolysate, and later differentiated into embryoids when transferred to MS agar medium with 0.1 mg/L zeatin. Eventually, embryoids developed into whole plantlets on the MS solid medium without phytohormones.     

从石刁柏原生质体培养再生植株

石刁柏,又名芦笋(Asparagus officinalisL.)是百合科天门冬属植物。其栽培品种含有丰富的维生素类及蛋白质。同时,石刁柏对于某些疾病有一定的药效,因此它已成为人们所喜爱的一种高级营养蔬菜。国外已有不少关于石刁柏试管苗繁殖的报告,但至今只有Bui Dang Ha等从石刁柏枝状叶分离的原生质体得到愈伤组织,并由此愈伤组织诱导获得了再生植株。此后,未见在石刁柏的原生质体培养方面再有新的工作。在本文中,我们利     

Plant Regeneration from Protoplasts of Coriandrum sativum

Calli produced from stem segments of seedling of Coriandrum satwum which were cultured on MS agar medium containing NAA 1.0mg/L. The embryogenic cell colony suspension was estabilished on MS liquid medium containing NAA 1.0mg/L%2,4-D 0.2mg/L+BA 0.5 mg/L. The cell suspension culture was used for protoplast preparation. Protoplasts were obtained in the enzyme mixture containing 2.0% Onozuka R-10, 1.0% pectinase, 0.5% snailase, 0.5% dextran sulfate potassium Salt, 0.6mol/L mannital CPW solution at pH 5.8 and 25℃. Cultured in a KM8P liquid medium containing NAA 1.0mg/L+2,4-D 0.2mg/L+6-BA 0.5 mg/L, glucose 0.4mol/L and CM 20mi/L; the protoplasts entered the stage of derision after three days, cell clusters formed in 10 days and calli formed after about 50 days. When the calli were transferred to MS agar medium containing many growth substances, they differentiated into embryoids, and then developed into plantlet with many green leaves and roots on the 1/2 MS agar medium.     

Induction of direct somatic organogenesis in onion (Allium cepa L.) using a two-step flower or ovary culture

A novel method for direct organogenesis in onion (Allium cepa L.) resulting in the formation of multiple shoot structures induced on mature flower buds or ovaries in a two-step culture procedure is described. Flowers were cultured on an induction medium containing 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 2 mg/l 6-benzylaminopurine (BAP). After 6 days (superior to 3 or 12 days), flowers or extracted ovaries were transferred to a differentiation medium containing 2 mg/l thidiazuron (TDZ). Medium solidification with gellan gum was superior to agar or agar/gellan gum mixture. A similar regeneration frequency was achieved at high (100 g/l) and lower (50 g/l) sucrose content. Regeneration was obtained from all 12 cultivars or inbred lines examined, although the efficiency and the occurrence of hyperhydricity varied depending on genotype and procedure used. Studies of plant growth regulators revealed that in the induction medium, the auxin 2,4-D was superior to 5 mg/l naphthaleneacetic acid or picloram, which partially or completely inhibited regeneration. Omitting cytokinin in the induction medium or substitution of BAP with 2 mg/l 2iP lowered regeneration, while substitution with 1 mg/l TDZ was equally effective. In the differentiation medium, lower concentrations of TDZ (1 and 0.5 mg/l) or substitution of TDZ with 5 mg/l BAP were equally or less effective. Received: 14 October 1998 / Revision received: 19 November 1998 / Accepted: 30 November 1998     

Plant regeneration from mesophyll protoplasts ofDianthus superbus

Leaf mesophyll protoplasts ofDianthus superbus were cultured at a density of 5 × 10⁴ protoplasts/ml and divided at about 18% plating efficiency in MS liquid medium supplemented with 0.5 mg/L BAP, 2.0 mg/L NAA and 9% mannitol after 2 weeks. Protocolonies formed after 3 to 4 weeks of culture in the dark at 27°C. These colonies were transferred to continuous illumination (21.5 E m^–2 sec^–1) for 2 weeks where most of the colonies divided to form microcalli, about 2 mm in diameter. Subsequently, green microcalli were transferred to MS solidified medium with 2.0 mg/L 2,4-D that induced shoot-forming calli after 4 weeks. These calli were transferred onto N₆-2 medium containing 0.1 mg/L 2,4-D, 0.1 mg/L NAA, 2.0 mg/L kinetin and 2.0 g/L casein hydrolysate and were cultured under light. After 5 weeks the calli gave rise to multiple shoots (10 to 15 per callus). Upon transfer to MS medium containing 2.0 mg/L NAA, individual shoots were rooted in 4 weeks. The regenerants were successfully transplanted into potting soil.Abbreviations MS Murashige and Skoog - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - N₆ Chu basal salt mixture - MES 2-N-morpholinoethanesulfonic acid     

Plant regeneration from cell suspension-derived protoplasts of Primula malacoides and Primula obconica

Protoplasts were isolated from cell suspension cultures of Primula malacoides cv. ‘Lovely Tokyo’ and P. obconica cv. ‘Aalsmeer Giant White’. P. obconica protoplasts were embedded in 0.1% (w/v) gellan gum-solidified discs comprising MS medium supplemented with 3 mg/l of 2,4-D or picloram, 0.1 mg/l of zeatin, 0.2 M glucose and 0.2 M mannitol, and surrounded by a liquid medium of the same composition except for the addition of 0.1% (w/v) activated charcoal. The protoplasts formed visible colonies, which were transferred to the regeneration medium containing 30 g/l of sucrose, 0.1 mg/l of picloram and 2 mg/l of zeatin for shoot induction. P. malacoides protoplasts formed visible colonies when cultured in disc culture using 0.1% (w/v) gellan gum-solidified MS medium containing 5 mg/l of 2,4-D, 1 mg/l of NAA, 0.1 mg/l of zeatin and 0.4 M glucose. Small calli were transferred to MS medium supplemented with 5 mg/l of zeatin for shoot regeneration. The shoots of both species readily rooted on plant growth regulator-free 1/2 MS medium and successfully acclimatized to greenhouse conditions. The protoplast-derived plants showed some alterations in morphological characteristics from those of the in-vitro-germinated control plants.     

Efficient callus formation and plant regeneration of goosegrass [<Emphasis Type="Italic">Eleusine indica </Emphasis>(L.) Gaertn.]

Efficient methods in totipotent callus formation, cell suspension culture establishment and whole-plant regeneration have been developed for the goosegrass [ Eleusine indica (L.) Gaertn.] and its dinitroaniline-resistant biotypes. The optimum medium for inducing morphogenic calli consisted of N6 basal salts and B5 vitamins supplemented with 1-2 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D), 2 mg l(-1) glycine, 100 mg l(-1) asparagine, 100 mg l(-1) casein hydrolysate, 30 g l(-1) sucrose and 0.6% agar, pH 5.7. The presence of organogenic and embryogenic structures in these calli was histologically documented. Cell suspension cultures derived from young calli were established in a liquid medium with the same composition. Morphogenic structures of direct shoots and somatic embryos were grown into rooted plantlets on medium containing MS basal salts, B5 vitamins, 1 mg l(-1) kinetin (Kn) and 0.1 mg l(-1) indole-3-acetic acid (IAA), 3% sucrose, 0.6% agar, pH 5.7. Calli derived from the R-biotype of E. indica possessed a high resistance to trifluralin (dinitroaniline herbicide) and cross-resistance to a structurally non-related herbicide, amiprophosmethyl (phosphorothioamidate herbicide), as did the original resistant plants. Embryogenic cell suspension culture was a better source of E. indica protoplasts than callus or mesophyll tissue. The enzyme solution containing 1.5% cellulase Onozuka R-10, 0.5% driselase, 1% pectolyase Y-23, 0.5% hemicellulase and N(6) mineral salts with an additional 0.2 M KCl and 0.1 M CaCl(2) (pH 5.4-5.5) was used for protoplast isolation. The purified protoplasts were cultivated in KM8p liquid medium supplemented with 2 mg l(-1) 2,4-D and 0.2 mg l(-1) Kn.     

Plant Regeneration from Protoplasts of Ligusticurn wallichii Franch

The hypocotyls of the embryoid derived plantlets of Ligusticum wallichii Franch were used for protoplast preparation. Protoplasts were obtained with the enzyme mixture containing 1.5% Onozuka R-10, 0.3% Macerozyme R-10, 0.5% Snailase, 5 mmol/l CaCl2, 1 mmol/l KH2PO4, 0.6 mol/l manitol, at pH 5.6–5.8 and 27℃. Protoplasts were cultured in a modified MS liquid medium containing 1 mg/l 2,4-D + 0.5 mg/l 6- BA. The first divisions were found after twelve days, and the dividing cells formed cell colonies of 0.5–1 mm after about fourty days. When they were transferred to MS agar medium (with half quantity of macronutrients) supplemented with 2,4-D (0.5mg/l) and 6-BA(0.5mg/l), they grew into calli. At last, on the medium without any phytohormones, the growing calli differentiated embryoids which developed into plantlets with many green leaves and roots.     

Protoplast culture and plant regeneration from Brassica carinata Braun

Protoplasts isolated from hypocotyls of three-day-old seedlings of Brassica carinata (Braun) cv R-2128 were cultured in a modified Nitsch and Nitsch liquid medium containing 13% sucrose, 0.4% Ficoll, 0.25 mg/l BA, 0.5 mg/l NAA and 0.5 mg/l 2,4-D. The density of medium caused the protoplasts and the developing microcalli to float on the surface of the liquid medium whereas all debris and lysed cells sank to the bottom of the culture plate. After 4–6 weeks developing microcalli were approximately 0.5 mm in diameter and were transferred onto MS medium containing 3% sucrose, 0.4% agarose, 200 mg/l casein hydrolysate, 5 mg/l BA and 0.5 mg/l NAA, pH 5.7. Approximately 20% of the calli transferred to this medium produced plantlets.Abbreviations BA 6-Benzylaminopurine - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashiqe-Skoog     

Embryogenesis and plant regeneration from anther culture of bamboo (Sinocalamus latiflora (Munro) McClure)

Embryogenic callus was initiated from bamboo (Sinocalumus satiflora (Munro) McClure) anthers cultured on N₆ medium supplemented with 1 mg/l 2,4-D, 1 mg/l BA, 2 g/l charcoal, 0.8% agar (Sigma) and 9% sucrose. Anthers with microspores at miduninucleate to early-binucleate stages showed better rate of response for callus induction. Prolonged culture of these embryogenic calli on the original medium or subculture to an auxin-free medium resulted in embryoid formation and their subsequent germination to form rooted plantlets. Chromosome counts from root-tip cells of anther-derived plant indicated that they were haploid (N=36).Abbreviations N₆ Chu et al. (1975) - MS Murashige and Skoog (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - BA 6-benzylaminopurine     

Plant regeneration from protoplasts isolated from callus ofGentiana crassicaulis

Fast growing calli induced from hypocotyl segments ofGentiana crassicaulis were used for preparation of protoplasts. High yields of viable protoplasts were produced in an enzyme solution containing 1–2% cellulase, I% pecfinase, and 0.5% Hemicellulase. Protoplasts were cultured in KM8P medium containing 1 mg/l 2,4-D, 0.5 mg/l 6BA, 500 mg/l LH, 0.5 M glucose and 0.1 M mannitol by the solid-liquid dual layer culture method. First division occurred within 4–5 days of culture at a frequency of 17.8%. Sustained divisions led to callus formation. Periodically diluting the cultures with freshly prepared liquid medium containing 1% glucose was critical for colony formation. Protocolonies about 2 mm in size were transferred onto MS medium supplemented with 3 mg/l ZT, 2 mg/l 6BA, 1 mg/l GA₃, 1 mg/l NAA and 6% sucrose to obtain embryogenic calli. Plantlets were regenerated via somatic embryogenesis at high frequency on hormone-free MS Medium.Abbreviations 6BA 6-benzylaminopurine - NAA naphthaleneacetic acid - 2,4-D 2,4 - dichlorophenoxyacetic acid - ZT zeatin - GA₃ gibberellic acid - LH lactalbumin hydrolysate - MES 2-(N-morpholino)-ethane sulfonic acid - MS Murashige & Skoog's medium(1962)     

Stable transformation of sugarbeet protoplasts by electroporation

Conditions were optimized for the culture, antibiotic selection and stable transformation by electroporation of suspension culture protoplasts of sugarbeet,Beta vulgaris L.. Highest plating efficiencies (up to 65% at day 21) were obtained if protoplasts were cultured in PGO salts (de Greef and Jacobs, 1979) supplemented with 0.1 mg/1 2,4-D, 0.01 mg/l BAP and 9% mannitol, and in 0.6% agarose rather than in liquid medium. Sensitivity to kanamycin also depended on whether protoplasts were cultured in liquid or agarose medium. Stable transformation of protoplast-derived colonies, as determined by resistance to kanamycin and Southern blot analysis, was achieved by electroporation using both rectangular and exponentially-decaying pulses.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - npt-II neomycin phosphotransferase - EDTA ethylenediaminetetracetic acid - SDS sodium dodecyl sulphate     

Protoplast isolation,culture and regeneration from Egyptian cultures of Pea and Beans

Abstract

A protocol of protoplast isolation from Egyptian varieties of pea and bean is reported. Protoplast cultures were established from apical shoots of pea (Pisum sativum) and suspension cultures of bean (Phaseolus vulgaris). To isolate protoplasts of pea, apical shoot tissues were digested for 10 h using enzyme solution containing 1% pectinase, 0.5% cellulase, 0.5% hemicellulase, 10% mannitol and 0.1% CaCl₂-2H₂O. For protoplast isolation from suspension culture of bean, collected cells were incubated for 6 h in digestion solution containing 0.5% pectinase, 0.25% of each of cellulase and hemicellulase, 10% mannitol and 0.1% CaCl₂-2H₂O. Purified protoplasts were cultured in liquid culture medium. Microcalli were obtained after 30 days of culture. Calli colonies with a diameter of about 5 mm were developed after one month of culturing on solid B5 medium containing 2% sucrose, 2 g/l casein hydrolysate, 0.7% agar and supplemented with either 1 mg/l of each 2,4-D and kin in case of pea or 2 mg/l 2,4-D+0.5 mg/l kin in case of bean. Protoplast derived callus of pea was successfully differentiated into shoot and root, and highest frequency of shoot organogenesis was recorded on medium containing 0.5 mg/l NAA+2 mg/l BA. Protoplast derived callus of bean, on the other hand, gave rise to a high frequency of root formation when cultured on medium containing 1 mg/l NAA, but attempts to regenerate shoots from this callus was unsuccessfull.     

骆驼刺发根农杆菌转化系的原生质体培养和植株再生

从发根农杆菌A4转化的荒漠植物—璐驼刺毛状根愈伤组织中分离的原生质体培养的结果表明,酶解新转代7～10d的淡黄色松软愈伤组织,可获得大量有活力的原生质体。原生质体在附加有1．5mg．L-1 2,4．D、0．2mg．L-1 6．BA、0．3m01．L-1甘露醇、2％（W/V）蔗糖和500mg·L-1水解酪蛋白的DPD培养基中进行液体浅层培养可持续分裂。培养基的最适渗透压为（450±3）mOsm·kg-1,原生质体的最适植板密度为4×10^5个．mL-1。制备原生质体的愈伤组织以低温（4℃）预处理后,原生质体的产率和分裂频率均提高,分裂频率最高可达50％。原生质体分裂形成的愈伤组织转移在附加1-2mg．L-1 6-BA（或KT）和0．2mg·L-1NAA的MS培养基上培养后,可以分化并获得再生植株。纸电泳检测表明,原生质体再生的愈伤组织和分化植株仍然含有毛状根转化系的特异产物——冠瘿碱。     

绞股蓝悬浮细胞的原生质体再生植株

绞股蓝(Gynostemma pentaphyllum (Thumb)Mak．)是葫芦科多年生草本药用植物,现已得到广泛的开发利用,本文首次报道了绞股蓝悬浮细胞的原生质体再生植株。